Synthesis, acidity and antioxidant properties of some novel 3,4-disubstituted-4,5-dihydro-1H-1,2,4-triazol-5-one derivatives

Article abstract of DOI:10.3390/molecules13010107

3-Alkyl(aryl)-4-amino-4,5-dihydro-1H-1,2,4-triazol-5-ones 2a-g reacted with 4-diethylaminobenzaldehyde to afford the corresponding 3-alkyl(aryl)-4-(4- diethyl-aminobenzylidenamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones 3a-g. The acetylation reactions of compounds 3a-e were investigated and compounds 4a-e were thus obtained. The new compounds were characterized using IR, ¹H-NMR, ¹³C-NMR, UV and MS spectral data. In addition, the newly synthesized compounds 3a-g were titrated potentiometrically with tetrabutylammonium hydroxide in four non-aqueous solvents such as isopropyl alcohol, tert-butyl alcohol, acetone and N,N-dimethylformamide (DMF), and the half-neutralization potential values and the corresponding pK_a values were determined for all cases. Moreover, 3 and 4 type compounds were also screened for their antioxidant activities.

Full text of DOI:10.3390/molecules13010107

Molecules 2008, 13, 107-121
molecules
ISSN 1420-3049
© 2008 by MDPI
www.mdpi.org/molecules
Full Paper
Synthesis, Acidity and Antioxidant Properties of Some Novel
3,4-disubstituted-4,5-dihydro-1H-1,2,4-triazol-5-one Derivatives
Muzaffer Alkan 1,*, Haydar Yüksek 2, Özlem Gürsoy-Kol 2 and Mustafa Calapoğlu 1
1 Education Faculty, Kafkas University, 36100 Kars, Turkey
2
Department of Chemistry, Kafkas University, 36100 Kars, Turkey; E-mail: hyuksek61@gmail.com
(Yüksek), ozlemgursoy@gmail.com (Gürsoy-Kol), mustafacalapoglu@yahoo.com (Calapoğlu)
* Author to whom correspondence should be addressed; E-mail: muzzo_61@yahoo.com; Fax (+90)-
474-2121185
Received: 6 December 2007; in revised form: 18 January 2008 / Accepted: 18 January 2008 /
Published: 19 January 2008
Abstract: 3-Alkyl(aryl)-4-amino-4,5-dihydro-1H-1,2,4-triazol-5-ones 2a-g reacted with
4-diethylaminobenzaldehyde to afford the corresponding 3-alkyl(aryl)-4-(4-diethyl-
aminobenzylidenamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones 3a-g. The acetylation
reactions of compounds 3a-e were investigated and compounds 4a-e were thus obtained.
1
The new compounds were characterized using IR, H-NMR, 13C-NMR, UV and MS
spectral data. In addition, the newly synthesized compounds 3a-g were titrated
potentiometrically with tetrabutylammonium hydroxide in four non-aqueous solvents such
as isopropyl alcohol, tert-butyl alcohol, acetone and N,N-dimethylformamide (DMF), and
the half-neutralization potential values and the corresponding pKa values were determined
for all cases. Moreover, 3 and 4 type compounds were also screened for their antioxidant
activities.
Keywords: 4,5-Dihydro-1H-1,2,4-triazol-5-ones, Schiff base, Acetylation, Antioxidant
activity, pKa, Potentiometric titrations.

Molecules 2008, 13
108
Introduction
1,2,4-Triazole and 4,5-dihydro-1H-1,2,4-triazol-5-one derivatives are reported to possess a broad
spectrum of biological activities such as antifungal, antimicrobial, hypoglycemic, antihypertensive,
analgesic, antiparasitic, hypocholesteremic, antiviral, anti-inflammatory, antitumor, antioxidant and
anti-HIV properties [1-7]. In addition, several articles reporting the synthesis of some
N-arylidenamino-4,5-dihydro-1H-1,2,4-triazol-5-one derivatives have been published [6-17]. The
acylation of 4,5-dihydro-1H-1,2,4-triazol-5-one derivatives has also been reported [3,5,9-12,17].
On the other hand, it is known that 1,2,4-triazole and 4,5-dihydro-1H-1,2,4-triazol-5-one rings
have weak acidic properties, so some 1,2,4-triazole and 4,5-dihydro-1H-1,2,4-triazol-5-one derivatives
were titrated potentiometrically with tetrabutylammonium hydroxide (TBAH) in non-aqueous
solvents, and the pKa values of the compounds were determined [6,7,9-14,18-21].
Furthermore, antioxidants are extensively studied for their capacity to protect organisms and cells
from damage induced by oxidative stress. Scientists in various disciplines have become more
interested in new compounds, either synthesized or obtained from natural sources, that could provide
active components to prevent or reduce the impact of oxidative stress on cells [22]. Exogenous
chemicals and endogenous metabolic processes in human body or in food system might produce highly
reactive free radicals, especially oxygen derived radicals, which are capable of oxidizing biomolecules,
resulting in cell death and issue damage. Oxidative damages play a significant pathological role in
human diseases. For example, cancer, emphysema, cirrhosis, atherosclerosis and arthritis have all been
correlated with oxidative damage. Also, excessive generation of ROS (reactive oxygen species)
induced by various stimuli and which exceeds the antioxidant capacity of the organism leads to a
variety of pathophysiological processes such as inflammation, diabetes, genotoxicity and cancer [23].
In the present paper, the antioxidant activities of seven new 3-alkyl(aryl)-4-(4-diethylamino-
benzylideneamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones 3a-g, which were synthesized by the reactions
of 3-alkyl-4-amino-4,5-dihydro-1H-1,2,4-triazol-5-ones 2a-g with 4-diethylaminobenzaldehyde and
five new 1-acetyl-3-alkyl(aryl)-4-(4-diethylaminobenzylideneamino)-4,5-dihydro-1H-1,2,4-triazol-5-
ones 4a-e, which were synthesized by the acetylations of 3a-e were determined (Scheme 1).
Scheme 1.
O
R
N
NH
N
NH
N
N
i
ii
iii
C
NNHCO2C2H5
O
O
O
N
N
N
N
N
R
R
R
OC2H5
NH2
N
N
2
3
1
4
i) N2H4, H2O, reflux, 6 h; ii) Et2NC6H4CHO (p-), AcOH, reflux, 1 h; iii) Ac2O, reflux, 1 h
a) R= CH3, b) R= CH2CH3, c) R= CH2C6H5, d) R= CH2C6H4.CH3 (p-), e) R= CH2C6H4.Cl
(p-), f) R= C6H5, g) R= cyclopropyl

Molecules 2008, 13
109
Moreover, we also examined the potentiometric titrations of the synthesized compounds 3 with
tetrabutylammonium hydroxide (TBAH) in four non-aqueous solvents (isopropyl alcohol, tert-butyl
alcohol, acetone and DMF) to determine the corresponding half-neutralization potentials (HNP) and
the corresponding pKa values. The data obtained from the potentiometric titrations was interpreted, and
the effect of the C-3 substituent in 4,5-dihydro-1H-1,2,4-triazol-5-one ring as well as solvent effects
were studied [9-14,18-21,24].
Results and Discussion
In this study, the structures of seven new 3-alkyl(aryl)-4-(4-diethylaminobenzylideneamino)-4,5-
dihydro-1H-1,2,4-triazol-5-ones 3a-g and five new 1-acetyl-3-alkyl(aryl)-4-(4-diethylamino-
1
benzylideneamino)-4,5-dihydro-1H-1,2,4-triazol-5-ones 4a-e were identified using IR, H-NMR,
13C- NMR, UV and MS spectral data. In addition, the compounds 3a-g and 4a-e were screened for
their in-vitro antioxidant activities. Several methods are used to determine antioxidant activities. The
methods used in this study are discussed below:
Total reductive capability using the potassium ferricyanide reduction method
The reductive capabilities of compounds are assessed by the extent of conversion of the
Fe3+/ferricyanide complex to the Fe2+/ferrous form. The reducing powers of the compounds were
observed at different concentrations, and results were compared with BHA, BHT and α-tocopherol.
The reducing capacity of a compound may serve as a significant indicator for its potential antioxidant
activity [25]. The antioxidant activity of a putative antioxidant has been attributed to various
mechanisms, among which are prevention chain initiation, binding of transition metal ion catalyst,
decomposition of peroxides, prevention of continued hydrogen abstraction, reductive capacity and
radical scavenging [26]. In this study, the reducing ability of compounds synthesized augmented with
increasing concentration of samples. As can be seen in Figure 1, compound 3g showed a moderate
reducing activity for Fe3+ compared to the blank reaction. Compounds 4a and 4b also displayed a weak
reducing activity. The other compounds showed lower absorbance than the blank, hence, no reductive
activities were observed. Only compounds 3g, 4a and 4b may reduce metal ions complexes to their
lower oxidation state or to take part in electron transfer reaction. In other words, these compounds
showed the ability of electron donor to scavenge free radicals.
DPPH• radical scavenging activity
The scavenging of the stable DPPH radical model is a widely used method to evaluate antioxidant
activities in a relatively short time compared with other methods. The effect of antioxidants on DPPH
radical scavenging was thought to be due to their hydrogen donating ability [27]. DPPH is a stable free
radical and accepts an electron or hydrogen radical to become a stable diamagnetic molecule [28]. The
reduction capability of DPPH radicals was determined by decrease in its absorbance at 517 nm
induced by antioxidants. The absorption maximum of a stable DPPH radical in ethanol was at 517 nm.

Molecules 2008, 13
Figure 1. Total reductive potential of different concentrations (50-100-150 µg/mL) of 3g,
110
4a, 4b, BHT and α-tocopherol. High absorbance at 700 nm indicates high reducing
power.
2
1,8
1,6
3g
1,4
4a
1,2
4b
1
BHT
0,8
BHA
0,6
α-Tocopherol
0,4
0,2
0
0
20
40
60
80
100
120
140
160
Concentration (µg\mL)
Figure 2. Scavenging effect of compounds 3a-g, BHA and α-tocopherol at different
concentrations (12,5-25-37.5 µg/mL).
120
100
80
3a
3b
3c
3d
60
40
20
0
3e
3f
3g
BHA
α-Tocopherol
0
5
10
15
20
25
30
35
40
Concentration (µg\mL)
The decrease in absorbance of DPPH radical was caused by antioxidants, because of reaction
between antioxidant molecules and radical, progresses, which result in the scavenging of the radical by
hydrogen donation. It is visually noticeable as a discoloration from purple to yellow. Hence, DPPH is
usually used as a substrate to evaluate antioxidative activity of antioxidants [29]. BHA and α-
tocopherol were used as a reference to antioxidant compounds. All the compounds tested with this
method exhibited marked DPPH free radical scavenging activity in a concentration-dependent manner.
Figures 2 and 3 illustrate a decrease in the concentration of DPPH radical due to the scavenging ability
of these compounds. These results indicate that the newly synthesized compounds showed moderate
activities as a radical scavenger, indicating that it has good activities as hydrogen donors.

Molecules 2008, 13
Figure 3. Scavenging effect of compounds 4a-e, BHA and α-tocopherol at different
111
concentrations (12,5-25-37.5 µg/mL).
120
100
80
60
40
20
0
4a
4b
4c
4d
4e
BHA
α-Tocopherol
0
5
10
15
20
25
30
35
40
Concentration (µg\mL)
Ferrous ion chelating activity
The chelating effect towards ferrous ions by the compounds and standards was determined
according to the method of Dinis [30]. Ferrozine can quantitatively form complexes with Fe2+. In the
presence of chelating agents, the complex formation is disrupted with the result that the red colour of
the complex is decreased. Measurement of colour reduction therefore allows estimation of the
chelating activity of the coexisting chelator [31]. Transition metals have pivotal role in the generation
oxygen free radicals in living organism. The ferric iron (Fe3+) is the relatively biologically inactive
form of iron. However, it can be reduced to the active Fe2+, depending on condition, particularly pH
[32] and oxidized back through Fenton type reactions with the production of hydroxyl radical or
Haber-Weiss reactions with superoxide anions. The production of these radicals may lead to lipid
peroxidation, protein modification and DNA damage. Chelating agents may not activate metal ions
and potentially inhibit the metal-dependent processes [33]. Also, the production of highly active ROS
.-
such as O2 , H2O2 and OH. is also catalyzed by free iron though Haber-Weiss reactions:
O.2 + H2O2 → O2 + OH- + OH.
Among the transition metals, iron is known as the most important lipid oxidation pro-oxidant due
to its high reactivity. The ferrous state of iron accelerates lipid oxidation by breaking down the
hydrogen and lipid peroxides to reactive free radicals via the Fenton reactions:
Fe2+ + H2O2 → Fe3+ + OH- + OH.
Fe3+ ion also produces radicals from peroxides, although the rate is tenfold less than that of Fe2+
ion, which is the most powerful pro-oxidant among the various types of metal ions [34]. Ferrous ion
chelating activities of the compounds, BHT and α-tocopherol are shown in Figure 4. In this study,
metal chelating capacity was significant since it reduced the concentrations of the catalyzing transition
metal. It was reported that chelating agents that form σ-bonds with a metal are effective as secondary
antioxidants because they reduce the redox potential thereby stabilizing the oxidized form of metal ion

Molecules 2008, 13
112
[35]. The data obtained from Figures 4 and 5 reveal that the compounds demonstrate a marked
capacity for iron binding, suggesting that their action as peroxidation protectors may be related to their
iron binding capacity. On the other hand, free iron is known to have low solubility and a chelated iron
complex has greater solubility in solution, which can be contributed solely by the ligand. Furthermore,
the compound-iron complex may also be active, since it can participate in iron-catalyzed reactions.
Figure 4. Metal chelating effect of different amount of the compounds 3a-g, BHT and α-
tocopherol on ferrous ions.
70
3a
60
3b
50
40
30
20
10
0
3c
3d
3e
3f
3g
BHT
α-Tocopherol
0
5
10
15
20
25
30
35
40
Concentration (µg\mL)
Figure 5. Metal chelating effect of different amount of the compounds 4a-e, BHT and α-
tocopherol on ferrous ions.
60
50
40
4a
4b
4c
4d
30
20
10
0
4e
BHT
α-Tocopherol
0
5
10
15
20
25
30
35
40
Concentration (µg\mL)
In conclusion, the data here reported could be of the possible interest because of the observed
hydrogen donating, radical scavenging and metal chelating activities of yhr dtudied compounds could
prevent redox cycling. Design and synthesis of novel small molecules which can specifically
protective role in biological systems are in perspective in modern medicinal chemistry.

Molecules 2008, 13
113
Potentiometric titrations
As a separate study, newly synthesized compounds 3 were titrated potentiometrically with TBAH
in four non-aqueous solvents: isopropyl and tert-butyl alcohol, acetone and DMF. The mV values read
in each titration were plotted against 0.05 M TBAH volumes (mL) added, and potentiometric titration
curves were obtained for all the cases. From the titration curves, the HNP values were measured, and
the corresponding pKa values were calculated. As an example, the potentiometric titration curves for
0.001 M solutions of compounds 3a-g titrated with 0.05 M TBAH in isopropyl alcohol are shown in
Figure 6.
Figure 6. Potentiometric titration curves of 0.001 M solutions of compound 3a-g titrated
with 0.05 M TBAH in acetone at 25°C.
-250
-300
-350
-400
-450
-500
-550
mL
0
10
20
3c
30
3e
3a
3b
3d
3f
The HNP values and the corresponding pKa values of compounds 3a-g, obtained from the
potentiometric titrations with 0.05 M TBAH in isopropyl alcohol, tert-butyl alcohol, acetone and
DMF, are presented in Table 1.
Table 1. The HNP and the corresponding pKa values of compounds 3a-g in isopropyl
alcohol, tert-butyl alcohol, acetone and DMF.
Isopropyl
alcohol
tert-butyl
DMF
Acetone
alcohol
Comp
d
HNP
HNP
HNP
HNP
pKa
pKa
pKa
pKa
(mV)
-300
-362
-336
-375
-313
-328
-
(mV)
-445
-473
-454
-473
-489
-403
-556
(mV)
-477
-387
-373
-507
-482
-432
-575
(mV)
-493
-483
-557
-583
-428
-258
-488
3a
3b
3c
3d
3e
3f
12.45
13.87
13.30
14.06
12.75
12.99
-
14.91
15.36
15.46
15.86
15.97
14.27
16.02
15.49
14.30
13.97
16.52
15.89
14.72
17.06
15.69
15.49
17.36
17.89
14.88
11.75
15.26
3g

Molecules 2008, 13
114
When the dielectric permittivity of solvents is taken into consideration, the acidity order can be
given as follows: DMF (ε=36.7) > acetone (ε=36) > isopropyl alcohol (ε=19.4) > tert-butyl alcohol
(ε=12). As seen in Table 1, the acidity order for compounds 3a and 3d is: isopropyl alcohol >
tert-butyl alcohol > DMF > acetone; for compounds 3b and 3c it is: isopropyl alcohol > DMF >
tert-butyl alcohol > acetone; for compound 3e it is: isopropyl alcohol > acetone > DMF > tert-butyl
alcohol and for compound 3f, it is: acetone > isopropyl alcohol > tert-butyl alcohol > DMF, while the
ranking for compound 3g is: acetone > tert-butyl alcohol > DMF. In isopropyl alcohol, all these
compounds show the strongest acidic properties.
The degree to which a pure solvent ionizes was represented by its autoprotolysis constant, KHS.
2HS = H2S+ + S-
For the above reaction the constant is defined by
KHS = [H2S+][S-]
Autoprotolysis is an acid-base reaction between identical solvent molecules is which some act as
an acid and others as a base. Consequently, the extent of an autoprotolysis reaction depends both on
the intrinsic acidity and the instrinsic basicity of the solvent. The importance of the autoprotolysis
constant in titrations lies in its effect on the completeness of a titration reaction [36]. The exchange of
the pKa values with autoprotolysis constant and dielectric constant are given in Figure 7.
As it is well known, the acidity of a compound depends on some factors. The two most important
factors are the solvent effect and molecular structure [9-14,18-21,24]. Table 1 and Figure 6 show that
the HNP values and corresponding pKa values obtained from the potentiometric titrations depend on
the non-aqueous solvents used and the substituents at C-3, in 4,5-dihydro-1H-1,2,4-triazol-5-one ring.
Figure 7. The variation of the pKa values for synthesized componds 3a-g with auto-
protolysis constant and dielectric constant.
3a
3b
3c
20
Dielectric Constant
3d
3e
3f
Autoprotolysis constant
18
16
14
12
10
3g

Molecules 2008, 13
115
Experimental
General
Melting points were taken on an Electrothermal 9100 digital melting point apparatus and are
uncorrected. IR spectra were registered on a Perkin-Elmer Spectrum One FT-IR spectrometer. 1H- and
13C-NMR spectra were recorded in deuterated dimethyl sulfoxide with TMS as internal standard on a
Varian Mercury spectrometer at 200 MHz and 50 MHz, respectively. UV absorption spectra were
measured in 10-mm quartz cells between 200 and 400 nm using a Unicam UV/VIS spectrometer.
Extinction coefficients (ε) are expressed in L·mol-1·cm-1. The starting compounds 2a-g were prepared
from the reactions of the corresponding ester ethoxycarbonylhydrazones 1a-g with an aqueous solution
of hydrazine hydrate as described in the literature [17,37].
General Method for the Preparation of 3-alkyl(aryl)-4-(4-diethylaminobenzylideneamino)-4,5-
dihydro-1H-1,2,4-triazol-5-ones 3
The corresponding compound 2 (0.01 mol) was dissolved in acetic acid (15 mL) and treated with
4-diethylaminobenzaldehyde (1.77 g, 0.01 mol). The mixture was refluxed for 1 h and then evaporated
at 50-55 °C in vacuo. Several recrystallizations of the residue from an appropriate solvent gave pure
compounds 3a-g as colourless crystals.
3-Methyl-4-(4-diethylaminobenzylideneamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (3a). Yield 84 %;
1
mp. 211 °C (EtOH); H-NMR: δ 1.09 (t, 6H, 2CH2CH3), 2.20 (s, 3H, CH3), 3.37 (q, 4H, 2CH2CH3),
6.69 (d, 2H, J=9.2 Hz, Ar-H), 7.57 (d, 2H, J=8.8 Hz, Ar-H), 9.37 (s, 1H, N=CH), 11.66 (s, 1H, NH);
13C-NMR: δ 11.88 (CH3), 13.06 (2CH3), 44.47 (2CH2), 111.67 (2C), 120.17, 130.25 (2C), 144.76
(4-diethylaminophenyl carbons), 150.51 (triazole C3), 152.19 (N=CH), 156.10 (triazole C5); IR: 3180
(NH), 1688 (C=O), 1594 (C=N), 818 (1,4-disubstituted Ar) cm-1; UV λmax (ε): 357 (25324), 230
(9898), 207 (16875) nm; MS: m/z 273 (M+), 274 (M+1).
3-Ethyl-4-(4-diethylaminobenzylideneamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (3b). Yield 94 %;
mp. 186 °C (EtOH); 1H-NMR: δ 1.08 (t, 6H, 2CH2CH3), 1.17 (t, 3H, CH2CH3), 2.61 (q, 2H, CH2CH3),
3.37 (q, 4H, 2CH2CH3), 6.69 (d, 2H, J=9.2 Hz, Ar-H), 7.56 (d, 2H, J=8.8 Hz, Ar-H), 9.37 (s, 1H,
13
N=CH), 11.68 (s, 1H, NH); C-NMR: δ 10.79 (CH3), 13.05 (2CH3), 19.34 (CH2), 44.47 (2CH2),
111.69 (2C), 120.23, 130.20 (2C), 148.58 (4-diethylaminophenyl carbons), 150.50 (triazole C3),
152.33 (N=CH), 156.07 (triazole C5); IR: 3179 (NH), 1683 (C=O), 1613, 1589 (C=N), 818
(1,4-disubstituted Ar) cm-1; UV λmax (ε): 358 (33660), 229 (12280), 207 (22775) nm.
3-Benzyl-4-(4-diethylaminobenzylideneamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (3c). Yield 97 %;
1
mp. 202 °C (EtOH); H-NMR: δ 1.09 (t, 6H, 2CH2CH3), 3.98 (s, 2H, CH2), 3.37 (q, 4H, 2CH2CH3),
6.69 (d, 2H, J=8.8 Hz, Ar-H), 7.19-7.29 (m, 5H, Ar-H), 7.54 (d, 2H, J=8.8 Hz, Ar-H), 9.35 (s, 1H,
13
N=CH), 11.82 (s, 1H, NH); C-NMR: δ 13.05 (2CH3), 31.84 (CH2), 44.47 (2CH2), 111.70 (2C),
120.16, 130.25 (2C), 146.78 (4-diethylaminophenyl carbons), 127.34, 129.09 (2C), 129.46 (2C),

Molecules 2008, 13
116
136.68 (phenyl carbons), 150.53 (triazole C3), 152.18 (N=CH), 155.74 (triazole C5); IR: 3165 (NH),
1700 (C=O), 1610, 1588 (C=N), 817 (1,4-disubstituted Ar), 770 and 683 (monosubstituted Ar) cm-1;
UV λmax (ε): 358 (17280), 209 (19526) nm.
3-(4-Methylbenzyl)-4-(4-diethylaminobenzylideneamino)-4,5-dihydro-1H-1,2,4-triazol-5-one
(3d).
1
Yield 94 %; mp. 198 °C (EtOH); H-NMR: δ 1.09 (t, 6H, 2CH2CH3), 2.22 (s, 3H, CH3), 3.92 (s, 2H,
CH2), 3.37 (q, 4H, 2CH2CH3), 6.69 (d, 2H, J=8.8 Hz, Ar-H), 7.07 (d, 2H, J=8.1 Hz, Ar-H), 7.17 (d,
13
2H, J=8.1 Hz, Ar-H), 7.55 (d, 2H, J=8.8 Hz, Ar-H), 9.35 (s, 1H, N=CH), 11.79 (s, 1H, NH); C-
NMR: δ 13.07 (2CH3), 21.78 (CH3), 31.43 (CH2), 44.47 (2CH2), 111.70 (2C), 120.20, 130.24 (2C),
146.93 (4-diethylaminophenyl carbons), 129.33 (2C), 129.65 (2C), 133.57, 136.36 (4-methylphenyl
carbons), 150.52 (triazole C3), 152.19 (N=CH), 155.68 (triazole C5); IR: 3173 (NH), 1702 (C=O),
1611, 1589 (C=N), 829, 817 (1,4-disubstituted Ar) cm-1; UV λmax (ε): 358 (30127), 210 (24382) nm.
3-(4-Chlorobenzyl)-4-(4-diethylaminobenzylideneamino)-4,5-dihydro-1H-1,2,4-triazol-5-one
(3e).
Yield 96 %; mp. 170 °C (EtOH); 1H-NMR: δ 1.09 (t, 6H, 2CH2CH3), 3.98 (s, 2H, CH2), 3.37 (q, 4H,
2CH2CH3), 6.68 (d, 2H, J=8.8 Hz, Ar-H), 7.32 (q, 4H, Ar-H), 7.53 (d, 2H, J=9.2 Hz, Ar-H), 9.36 (s,
1H, N=CH), 11.85 (s, 1H, NH); 13C-NMR: δ 13.07 (2CH3), 31.18 (CH2), 44.47 (2CH2), 111.70 (2C),
120.13, 130.27 (2C), 146.43 (4-diethylaminophenyl carbons), 129.02 (2C), 131.38 (2C), 132.05,
135.64 (4-chlorophenyl carbons), 150.54 (triazole C3), 152.17 (N=CH), 155.78 (triazole C5); IR: 3176
(NH), 1701 (C=O), 1602, 1587 (C=N), 846, 812 (1,4-disubstituted Ar) cm-1; UV λmax (ε): 360
(20757), 221 (13254), 210 (16274) nm.
3-Phenyl-4-(4-diethylaminobenzylideneamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (3f). Yield 84 %;
1
mp. 232 °C (EtOH); H-NMR: δ 1.08 (t, 6H, 2CH2CH3), 3.36 (q, 4H, 2CH2CH3), 6.70 (d, 2H, J=8.8
Hz, Ar-H), 7.47-7.48 (m, 3H, Ar-H), 7.56-7.59 (m, 2H, Ar-H), 7.90 (d, 2H, J=9.2 Hz, Ar-H), 9.24 (s,
1H, N=CH), 12.24 (s, 1H, NH); 13C-NMR: δ 13.05 (2CH3), 44.50 (2CH2), 111.75 (2C), 119.90,
130.56 (2C), 144.99 (4-diethylaminophenyl carbons), 127.10, 128.35 (2C), 129.19 (2C), 130.56
(phenyl carbons), 150.73 (triazole C3), 152.35 (N=CH), 159.33 (triazole C5); IR: 3160 (NH), 1687
(C=O), 1609, 1588 (C=N), 812 (1,4-disubstituted Ar), 769 and 690 (monosubstituted Ar) cm-1; UV
λ
max (ε): 364 (36264), 240 (21813), 210 (25875) nm.
3-Cyclopropyl-4-(4-diethylaminobenzylidenamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (3g). Yield 52
1
%; mp. 194 °C (EtOH-water, 1:3); H-NMR: δ 0.85-0.98 (m, 4H, CH2CH2), 1.08 (t, 6H, 2CH2CH3),
1.99-2.06 (m, 1H, CH), 3.31-3.43 (m, 4H, 2CH2CH3), 6.65-6.71 (m, 2H, Ar-H), 7.58 (d, 2H, J=8.7 Hz,
Ar-H), 9.33 (s, 1H, N=CH), 11.63 (s, 1H, NH); IR: 3170 (NH), 1695 (C=O), 1598 (C=N), 818 (1,4-
disubstituted Ar) cm-1; UV λmax (ε): 359 (20400), 240 (6100) nm.
General Method for the Preparation of 1-Acetyl-3-alkyl(aryl)-4-(4-diethylaminobenzylideneamino)-
4,5-dihydro-1H-1,2,4-triazol-5-ones 4
The corresponding compound 3 (0.01 mol) was refluxed with acetic anhydride (15 mL) for 0.5 h.
After addition of absolute ethanol (50 mL), the mixture was refluxed for 1 h more. Evaporation of the

Molecules 2008, 13
117
resulting solution at 40-45 °C in vacuo and several recrystallizations of the residue from EtOH gave
pure compounds 4a-e as colourless crystals.
1-Acetyl-3-methyl-4-(4-diethylaminobenzylideneamino)-4,5-dihydro-1H-1,2,4-triazol-5-one
(4a).
1
Yield 94 %; mp. 147 °C (EtOH); H-NMR: δ 1.09 (t, 6H, 2CH2CH3), 2.26 (s, 3H, CH3), 2.44 (s, 3H,
COCH3), 3.38 (q, 4H, 2CH2CH3), 6.70 (d, 2H, J=8.8 Hz, Ar-H), 7.59 (d, 2H, J=8.4 Hz, Ar-H), 9.17 (s,
13
1H, N=CH); C-NMR: δ 11.97 (CH3), 13.04 (2CH3), 24.11 (COCH3), 44.51 (2CH2), 111.67 (2C),
119.44, 130.69 (2C), 147.36 (4-diethylaminophenyl carbons), 148.83 (triazole C3), 150.92 (N=CH),
158.75 (triazole C5), 166.68 (COCH3); IR: 1768, 1696 (C=O), 1597, 1588 (C=N), 836 (1,4-
disubstituted Ar) cm-1; UV λmax (ε): 361 (22015), 230 (9972), 209 (14910) nm; MS: m/z 315 (M+),
316 (M+1).
1-Acetyl-3-ethyl-4-(4-diethylaminobenzylideneamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (4b). Yield
1
85 %; mp. 127 °C (EtOH); H-NMR: δ 1.06-1.23 (m, 9H, 3CH2CH3), 2.45 (s, 3H, COCH3), 2.64 (q,
2H, CH2CH3), 3.39 (m, 4H, 2CH2CH3), 6.69 (d, 2H, J=8.8 Hz, Ar-H), 7.57 (d, 2H, J=8.1 Hz, Ar-H),
13
9.16 (s, 1H, N=CH); C-NMR: δ 9.33 (CH3), 12.25 (2CH3), 18.61 (CH2), 23.35 (COCH3), 43.73
(2CH2), 110.85 (2C), 118.64, 129.86 (2C), 150.09 (4-diethylaminophenyl carbons), 148.26 (triazole
C3), 150.10 (N=CH), 157.89 (triazole C5), 165.88 (COCH3); IR: 1765, 1693 (C=O), 1605, 1586
(C=N), 835 (1,4-disubstituted Ar) cm-1; UV λmax (ε): 361 (22862), 232 (9455), 209 (13357) nm; MS:
m/z 329 (M+), 330 (M+1).
1-Acetyl-3-benzyl-4-(4-diethylaminobenzylideneamino)-4,5-dihydro-1H-1,2,4-triazol-5-one (4c). Yield
1
91 %; mp. 146 °C (EtOH); H-NMR: δ 1.09 (t, 6H, 2CH2CH3), 2.47 (s, 3H, COCH3), 4.06 (s, 2H,
CH2), 3.34-3.41 (m, 4H, 2CH2CH3), 6.71 (d, 2H, J=9.1 Hz, Ar-H), 7.26-7.33 (m, 5H, Ar-H), 7.57 (d,
13
2H, J=8.7 Hz, Ar-H), 9.16 (s, 1H, N=CH); C-NMR: δ 13.07 (2CH3), 24.25 (COCH3), 31.77 (CH2),
44.51 (2CH2), 111.69 (2C), 119.38, 130.74 (2C), 149.07 (4-diethylaminophenyl carbons), 127.63,
129.19 (2C), 129.69 (2C), 135.48 (phenyl carbons), 149.07 (triazole C3), 150.92 (N=CH), 158.45
(triazole C5), 166.73 (COCH3); IR: 1737, 1729 (C=O), 1604, 1585 (C=N), 820 (1,4-disubstituted Ar),
764 and 689 (monosubstituted Ar) cm-1; UV λmax (ε): 362 (28665), 235 (11040), 210 (22050) nm; MS:
m/z 391 (M+), 392 M+1).
1-Acetyl-3-(4-methylbenzyl)-4-(4-diethylaminobenzylideneamino)-4,5-dihydro-1H-1,2,4-triazol-5-one
(4d). Yield 91 %; mp. 142 °C (EtOH); 1H-NMR: δ 1.10 (t, 6H, 2CH2CH3), 2.23 (s, 3H, CH3), 2.47 (s,
3H, COCH3), 4.00 (s, 2H, CH2), 3.39 (q, 4H, 2CH2CH3), 6.71 (d, 2H, J=8.8 Hz, Ar-H), 7.09 (d, 2H,
J=7.7 Hz, Ar-H), 7.21 (d, 2H, J=8.1 Hz, Ar-H), 7.57 (d, 2H, J=8.8 Hz, Ar-H), 9.15 (s, 1H, N=CH);
13C-NMR: δ 13.07 (2CH3), 21.31 (CH3), 24.22 (COCH3), 31.39 (CH2), 44.51 (2CH2), 111.73 (2C),
119.45, 130.72 (2C), 149.02 (4-diethylaminophenyl carbons), 129.56 (2C), 129.74 (2C), 132.33,
136.71 (4-methylphenyl carbons), 149.20 (triazole C3), 150.20 (N=CH), 158.46 (triazole C5), 166.70
(COCH3); IR: 1737, 1728 (C=O), 1604, 1587 (C=N), 843, 820 (1,4-disubstituted Ar) cm-1; UV λmax
(ε): 362 (27633), 240 (9881), 209 (18927) nm.

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1-Acetyl-3-(4-chlorobenzyl)-4-(4-diethylaminobenzylideneamino)-4,5-dihydro-1H-1,2,4-triazol-5-one
(4e). Yield 88 %; mp. 164 °C (EtOH); 1H-NMR: δ 1.10 (t, 6H, 2CH2CH3), 2.47 (s, 3H, COCH3), 4.07
(s, 2H, CH2), 3.39 (q, 4H, 2CH2CH3), 6.71 (d, 2H, J=9.2 Hz, Ar-H), 7.36 (s, 4H, Ar-H), 7.56 (d, 2H,
J=8.8 Hz, Ar-H), 9.17 (s, 1H, N=CH); IR: 1739, 1701 (C=O), 1605, 1585 (C=N), 817, 802 (1,4-
disubstituted Ar) cm-1; UV λmax (ε): 363 (36264), 222 (21813), 210 (25875) nm.
Antioxidant Activity: Chemicals
Butylated hydroxytoluene (BHT) was purchased from E. Merck. Ferrous chloride, α-tocopherol,
1,1-diphenyl-2-picryl-hydrazyl (DPPH.), 3-(2-pyridyl)-5,6-bis(phenylsulfonic acid)-1,2,4-triazine
(ferrozine), butylated hydroxyanisole (BHA) and trichloracetic acid (TCA) were bought from Sigma
(Sigma –Aldrich GmbH, Sternheim,Germany).
Reducing power
The reducing power of the synthesized compounds was determined according to the method of
Oyaizu [38]. Different concentrations of the samples (50-250 µg/mL) in DMSO (1 mL) were mixed
with phosphate buffer (2.5 mL, 0.2 M, pH = 6.6) and potassium ferricyanide (2.5 mL, 1%). The
mixture was incubated at 50oC for 20 min. after which a portion (2.5 mL) of trichloroacetic acid (10%)
was added to the mixture, which was then centrifuged for 10 min at 1000 x g. The upper layer of
solution (2.5 mL) was mixed with distilled water (2.5 mL) and FeCI3 (0.5 mL, 0.1%), and then the
absorbance at 700 nm was measured in a spectrophometer. Higher absorbance of the reaction mixture
indicated greater reducing power.
Free radical scavenging activity
Free radical scavenging activity of compounds was measured by DPPH., using the method of Blois
[39]. Briefly, 0.1 mM solution of DPPH. in ethanol was prepared, and this solution (1 mL) was added
to sample solutions in DMSO (3 mL) at different concentrations (50-250 µg/mL). The mixture was
shaken vigorously and allowed to stand at room temperature for 30 min. Then the absorbance was
measured at 517 nm in a spectrophometer. Lower absorbance of the reaction mixture indicated higher
free radical scavenging activity. The DPPH. concentration (mM) in the reaction medium was
calculated from the following calibration curve and determined by linear regression (R: 0.997):
Absorbance = 0.0003 x DPPH. – 0.0174
The capability to scavenge the DPPH radical was calculated using the following equation:
DPPH. scavenging effect (%) = (A0 – A1/A0) x 100
where A0 is the absorbance of the control reaction and A1 is the absorbance in the presence of the
samples or standards.

Molecules 2008, 13
119
Metal chelating activity
The chelation of ferrous ions by the synthesized compounds and standards were estimated by the
method of Dinis et al. [30]. Briefly, the synthesized compounds (50-250 µg/mL) were added to a 2
mM solution of FeCI2 (0.05 mL). The reaction was initiated by the addition of 5 mM ferrozine (0.2
mL) and the mixture was shaken vigorously and left standing at room temperature for 10 min. After
the mixture had reached equilibrium, the absorbance of the solution was then measured at 562 nm in a
spectrophotometer. All test and analyses were run in triplicate and averaged. The percentage of
inhibition of ferrozine-Fe2+ complex formation was given by the formula: % Inhibition = (A0 – A1/A0)
x 100, where A0 is the absorbance of the control, and A1 is the absorbance in the presence of the
samples or standards. The control did not contain compound or standard.
Potentiometric Titrations
A Jenway 3040-model ion analyzer and an Ingold pH electrode were used for potentiometric
titrations. For each compound that would be titrated, the 0.001 M solution was separately prepared in
each non-aqueous solvent. The 0.05 M solution of TBAH in isopropyl alcohol, which is widely used in
the titration of acids, was used as titrant. The mV values, that were obtained in pH-meter, were
recorded. Finally, the HNP values were determined by drawing the mL (TBAH)-mV graphic.
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